Two-photon microscope is a powerful tool for in vivo imaging of cellular and extracellular matrix structures with sub-micron resolution. Two-photon based non-invasive optical biopsy methods have the potential of being used as an adjunct to excisional biopsy and histopathology. While the utility of two-photon microscopy for biological studies has been clearly demonstrated in areas such as neurobiology and embryology, its clinical potential remains unrealized. The operational complexity, size, imaging speed and cost stand in the way of testing this new technology in a clinical setting. This proposal addresses these difficulties by engineering a compact two-photon optical biopsy probe. A hand-held device will be built to image tissue cellular structures at video rate (> 10 frames/sec) with sub-cellular resolution down to a depth of 150-200 -1. A number of preliminary studies have been completed to demonstrate the feasibility of this project. (1) The use of two- photon excitation to image tissue cellular structures and metabolism based on its autofluorescence was demonstrated, Preliminary data indicating this technique's potential to distinguish normal and malignant tissues are included. (2) A prototype video-rate two-photon microscope was constructed. (3) A confocal reflected-light imaging sub-system was incorporated into a two-photon microscope. (4) The distribution of tissue biochemical constituents has been resolved based on their two-photon spectra. (5) The key two-photon photodamage mechanisms were identified, The aim of this proposal is to develop a hand-held two-photon biopsy probe suitable for clinical research. We will study the engineering challenges associated with miniaturizing two-photon technology such as the delivep ultra-short light pulses. It is also critical to avoid tissue photodamage; the maximum perr&sibl.e laser power and dosage levels will be established. We will characterize the performance this device in tissue phantoms, animal models and excised human skin biopsy specimens. We hope that the successful completion of this project will result in a first generation device that will allow the evaluation of two-photon optical biopsy techniques in the clinics.